HP-3228 and related peptides to treat sexual dysfunction

ABSTRACT

Methods for treating erectile dysfunction in males and sexual dysfunction, such as sexual arousal disorder, in females. The methods involve administering an effective dose of certain compounds, such as HP-228, having the generic formula 
     
       
         X 1 -X′ 2 -(D)Phe-Arg-(D)Trp-X 3 .

This application is a continuation-in-part of U.S. application Ser. No.09/306,686, filed May 6, 1999, which is a continuation of U.S.application Ser. No. 09/301,391, filed Apr. 28, 1999, now U.S. Pat. No.6,127,381, which claims benefit of U.S. Provisional Application No.60/083,368, filed Apr. 28, 1998, each of which is incorporated herein byreference. All publications and patents cited herein are alsoincorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to melanocortin receptors and morespecifically to the treatment of sexual dysfunction using melanocortinreceptor 3 ligands.

2. Background Information

Sexual dysfunction can be due to several physiological, as well aspsychological, factors. In males, erectile dysfunction can be associatedwith diseases such as diabetes mellitus, syphilis, alcoholism, drugdependency, hypopituitarism and hypothyroidism. Erectile dysfunction canalso be caused by vascular and neurogenic disorders, or be a side effectof drugs such as hypertensives, sedatives, tranquilizers andamphetamines. In all, erectile dysfunction is estimated to affect up to10 million men in the United States, with its incidence increasing withage up to 25% of men at age 65.

While various pharmaceutical treatments are commercially available orbeing developed, the underlying physiological bases for sexualdysfunction are not well understood. Attention has recently been drawnto melanocortin (MC) receptors, which are a group of cell surfaceproteins that mediate a variety of physiological effects. The MCreceptors have been implicated in the regulation of adrenal glandfunction such as production of the glucocorticoid cortisol andaldosterone, control of melanocyte growth and pigment production,thermoregulation, immunomodulation and analgesia. Five distinct MCreceptors have been cloned, although the specific role of each MCreceptor is still unclear.

Certain compounds, termed “melanocortins” have been found to bind MCreceptors, causing the activity of the receptors to increase ordecrease. These melanocortins include melanocyte-stimulating hormones(MSH) such as α-MSH, β-MSH and γ-MSH, as well as adrenocorticotropichormone (ACTH). Other compounds may bind as ligands to MC receptors,increasing or decreasing the activity of the receptors.

Thus, there is a need for compounds that can affect the activity ofspecific MC receptors that are involved with sexual dysfunction. Thepresent invention satisfies this need and provides related advantages aswell.

SUMMARY OF THE INVENTION

The present invention provides a method for treating sexual dysfunction,such as erectile dysfunction or sexual arousal disorder, with a compoundhaving the eneric formula X₁-X₂-(D)Phe-Arg-(D)Trp-X₃. A particularlyuseful compound is HP-228, which has the formulaAc-Nle-Gln-His-(D)Phe-Arg-(D)Trp-Gly-NH₂.

The invention also provides methods for selecting melanocortinreceptor-3 ligands by determining whether a compound modulates theactivity of MC-3 as an agonist or antagonist. These methods can be usedto screen compound libraries for ligands to treat sexual dysfunction andother MC-3-associated conditions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for treating sexual dysfunction.The term “sexual dysfunction” herein means any condition that inhibitsor impairs normal sexual function, including coitus. However, the termneed not be limited to physiological conditions, but may includepsychogenic conditions or perceived impairment without a formaldiagnosis of pathology.

In males, sexual dysfunction includes erectile dysfunction. The term“erectile dysfunction” or “impotence” means herein the inability orimpaired ability to attain or sustain an erection that would be ofsatisfactory rigidity for coitus. Sexual dysfunction in males can alsoinclude premature ejaculation and priapism, which is a condition ofprolonged and sometimes painful erection unrelated to sexual activity,often associated with sickle-cell disease.

In females, sexual dysfunction includes sexual arousal disorder. Theterm “sexual arousal disorder” means herein a persistent or recurrentfailure to attain or maintain the lubrication-swelling response ofsexual excitement until completion of sexual activity. Sexualdysfunction in females can also include inhibited orgasm anddyspareunia, which is painful or difficult coitus.

The method for treating sexual dysfunction comprises the step ofadministering to the subject an effective dose of the compound

X₁-X₂-(D)Phe-Arg-(D)Trp-X₃

wherein

R₁ is H, COCH₃, C₂H₅, CH₂Ph, COPh, COO-t-butyl, COOCH₂Ph,CH₂CO-(polyethylene glycol) or A;

R₂ is H, COCH₃, C₂H₅ or CH₂Ph;

R₃ is a linear alkyl group having 1 to 6 carbon atoms or a branched orcyclic alkyl group having 3 to 6 carbon atoms;

R₄ is (CH₂)_(m)—CONH₂, (CH₂)_(m)—CONHR₁ or (CH₂)_(m)—CONHA;

R₅ is OH, OR₃, NH₂, SH, NHCH₃, NHCH₂Ph or A;

R₆ is H or R₃;

R₇ is H, COCH₃, C₂H₅, CH₂Ph, COPh, COO-t-butyl, COOCH₂Ph orCH₂CO-(polyethylene glycol);

Ph is C₆H₅; m is 1, 2 or 3; n is 0, 1, 2 or 3; Y¹ and Y² areindependently hydrogen atoms, or are taken together to form a carbonylor thiocarbonyl; and A is

These peptide compounds are characterized in part by the core structures(D)Phe-Arg-(D)Trp or His-(D)Phe-Arg-(D)Trp and are described in U.S.Pat. No. 5,420,109, issued May 30, 1995, and U.S. Pat. No. 5,726,156,issued Mar. 10, 1998.

Particular compounds can be selected by further defining one or more ofthe individual variables in the generic formula above. The variable X₁can be norleucine or Ac-norleucine; or even norvaline, Ac-norvaline,leucine, Ac-leucine, isoleucine or Ac-isoleucine. The variable X₂ can beGln-His or His. The variable X₃ can be Gly or Gly-NH₂. The variable R₁can be H, C₂H₅ or CH₂Ph. The variables R₁ and R₂ can be COCH₃ or Hindependently. The variable R₅ can be NH₂. The variable R₅ can becovalently bound to X₁, forming a cyclic peptide.

A particularly useful compound is HP-228, which has the formulaAc-Nle-Gln-His-(D)Phe-Arg-(D)Trp-Gly-NH₂. HP-228 can be synthesized asdescribed in Example I.A. The effectiveness of HP-228 in treating sexualdysfunction such as erectile dysfunction is demonstrated in Example II.

Other useful compounds can have the (D)Phe of the formula be iodinatedin the para position. Thus, a useful compound is HP-467, which has theformula Ac-Nle-Gln-His-(para-iodo-(D)Phe)-Arg-(D)Trp-Gly-NH₂. HP-467 canbe prepared as further described in Example I.B.

Other specific compounds useful in the method of the invention include

(D) Phe-Arg-(D)Trp

Ac-(D)Phe-Arg-(D)Trp

(D)Phe-Arg-(D)Trp-NH₂

Ac-(D)Phe-Arg-(D)Trp-NH₂

(cyclohexyl)Gly-Gln-His-(D)Phe-Arg-(D)Trp-Gly

Ac-(cyclohexyl)Gly-Gln-His-(D)Phe-Arg-(D)Trp-Gly-NH₂

cyclo(His-(D)Phe-Arg-(D)Trp)

Nle-Gln-His-(D)Phe-Arg-(D)Trp-Gly-NH₂

His-(D)Phe-Arg-(D)Trp-Gly

His-(D)Phe-Arg-(D)Trp-Gly-NH₂

Ac-His-(D)Phe-Arg-(D)Trp-NH₂

His-(D)Phe-Arg-(D)Trp-OH

His-(D)Phe-Arg-(D)Trp

His-(D)Phe-Arg-(D)Trp-NH₂

Ac-His-(D)Phe-Arg-(D)Trp-OH

Ac-His-(D)Phe-Arg-(D)Trp-Gly-NH₂

Ac-Nle-Gln-His-(D)Phe-Arg-(D)Trp-Gly-OH

Ac-Nle-Gln-His-(D)Phe-Arg-(D)Trp-Gly-OC₂H₅

Ac-Nle-Gln-His-(D)Phe-Arg-(D)Trp-Gly-NH-NH₂

Ac-Nle-Asn-His-(D)Phe-Arg-(D)Trp-Gly-NH₂

Ac-Nle-Asn-His-(D)Phe-Arg-(D)Trp-Gly-OH

Ac-Nle-Gln-His-(D)Phe-Arg-(D)Trp-Gly-NHCH₂CH₂Ph

Ac-Nle-Gln-His-(D)Phe-Arg-(D)Trp-Gly-NHCH₂Ph

Gln-His-(D)Phe-Arg-(D)Trp-Gly-NH₂

Ac-Gln-His-(D)Phe-Arg-(D)Trp-Gly-NH₂

Ac-Nle-Gln-His-(D)Phe-Arg-(D)Trp-NH₂

Ac-His-(D)Phe-Arg-(D)Trp(CH₂)-(NAc)Gly-NH₂

His-(D)Phe-Arg-(D)Trp(CH₂)-(NAc)Gly

Amino acids are indicated by their commonly known three-letter code. Nleis the three-letter code for norleucine. The prefix (D)-designates anamino acid having the D-configuration, as opposed to the naturallyoccurring L-configuration. Where no specific configuration is indicated,a skilled artisan would understand the amino acid to be an L-amino acid.Finally, Ph indicates a phenyl group (C₆H₅).

A skilled artisan would know that the choice of amino acids or aminoacid analogs incorporated into the compound will depend in part on thespecific physical, chemical or biological characteristics required ofthe compound. Such characteristics can be determined by the route ofadministration and the desired location of action.

Selective modification of the reactive groups also can impart desirablecharacteristics to the compound. During synthesis, compounds can bemanipulated while still attached to a resin to obtain N-terminalmodified compounds such as an acetylated peptide or can be removed fromthe resin using hydrogen fluoride or an equivalent cleaving reagent andthen modified. Compounds synthesized containing the C-terminal carboxylgroup (Wang resin) can be modified after cleavage from the resin orprior to solution phase synthesis.

Methods for modifying the N-terminus or C-terminus of a peptide are wellknown in the art and include methods for acetylating the N-terminus oramidating the C-terminus. Similarly, methods for modifying side chainsof the amino acids or amino acid analogs are well known to those skilledin the art of peptide synthesis. The choice of modifications made to thereactive groups present on the peptide will be determined by the desiredcharacteristics.

Cyclic peptides can also be compounds useful in the method of theinvention. A cyclic peptide can be obtained by inducing the formation ofa covalent bond between the amino group at the N-terminus of the peptideand the carboxyl group at the C-terminus. For example, the peptidecyclo(His-(D)Phe-Arg-(D)Trp) can be produced by forming a covalent bondbetween His and (D)Trp. A cyclic peptide can also be obtained by forminga covalent bond between a terminal reactive group and a reactive aminoacid side chain or between two reactive amino acid side chains. Oneskilled in the art would know that the choice of a particular cyclicpeptide is determined by the reactive groups present on the peptide aswell as the desired characteristic of the peptide. For example, a cyclicpeptide may provide a compound with increased stability in vivo.

The compound can also be administered to the subject by any number ofroutes known in the art. These routes include injection parenterally,such as intravenously (i.v.) for systemic administration,intramuscularly, subcutaneously, intraorbitally, intracapsularly,intraperitoneally (i.p.), intracisternally, intra-articularly. Otherroutes include orally, intravaginally, rectally, or oral or topicalintubation, which can include direct application of an ointment orpowder, or using a nasal spray or inhalant, which may include apropellant.

The compound can be administered through the skin by passive absorptionsuch as a skin patch or facilitated absorption such as transdermaliontophoresis. Particular routes include transdermal delivery by passagethrough the skin into the blood stream and transmucosal delivery throughmucosal tissue. Another route is transurethral or intraurethral, wherethe compound contacts and passes through the wall of the urethra andenters the blood stream.

The compound can also be incorporated into liposomes, microspheres orother polymer matrices (Gregoriadis, Liposome Technology, Vols. I toIII, 2nd ed. (CRC Press, Boca Raton Fla. (1993)). Liposomes, whichconsist of phospholipids or other lipids, are nontoxic, physiologicallyacceptable and metabolizable carriers that are relatively simple to makeand administer.

An “effective dose” of the compound herein means an amount of thecomposition that is sufficient to therapeutically alleviate a sexualdysfunction in a subject or to prevent or delay onset or recurrence ofthe dysfunction.

The amount of a therapeutically effective dose depends on a variety offactors, including the particular characteristics of the compound, thetype and severity of the sexual dysfunction and the patient's medicalcondition. Based on such factors, a skilled physician can readilydetermine a therapeutically effective dose of the compound, which can beabout 0.01 to 100 mg/kg body weight per administration. For example, thecompound can be administered at 0.0001, 0.0005, 0.001, 0.005, 0.01,0.05, 0.1, 0.5, 1, 5, 10, 50 or 100 mg/kg body weight.

The total amount of compound can be administered as a single dose as abolus or by infusion over a relatively short period of time, or can beadministered in multiple doses administered over a more prolonged periodof time. One skilled in the art would know that the amount of a compounddepends on many factors including the age and general health of thesubject as well as the route of administration and the number oftreatments to be administered. In view of these factors, the skilledartisan would adjust the particular dose to obtain an effective dose fortreating sexual dysfunction.

A compound can be administered to a subject as a pharmaceuticalcomposition comprising the compound and a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers are well known in the artand include aqueous solutions such as physiologically buffered saline orother solvents or vehicles such as glycols, glycerol, oils such as oliveoil or injectable organic esters.

A pharmaceutically acceptable carrier can contain physiologicallyacceptable compounds that stabilize the compound or increase itsabsorption. Such physiologically acceptable compounds includecarbohydrates such as glucose, sucrose or dextrans; antioxidants, suchas ascorbic acid or glutathione; chelating agents; low molecular weightproteins or other stabilizers or excipients. One skilled in the artwould know that the choice of a pharmaceutically acceptable carrierdepends on the route of administration of the compound and on itsparticular physico-chemical characteristics.

The compounds described above can interact with melanocortin (MC)receptors to affect their activity. Five distinct MC receptors have beencloned and are known to mediate a variety of physiological effects. Forexample, MC-1 is involved in pain and inflammation. The MC receptorshave also been implicated in the regulation of weight control, adrenalgland function such as production of the glucocorticoid cortisol andaldosterone, control of melanocyte growth and pigment production,thermoregulation, immunomodulation and analgesia.

The diversity of physiological responses to MC receptor signaling can beused to alter or regulate a physiological pathway that mediates ormoderates a pathological condition or disease. Thus, the binding of anMC receptor ligand to an MC receptor can be used to modulatephysiological responses.

The recent elucidation of the role of various MC receptors in particularphysiological pathways supports the use of ligands that activatespecific MC receptors to modulate a physiological effect associated witha given condition or disease. The A-MSH analog MELANOTAN-II, which is anMC receptor ligand, has been shown to cause penile erections in humansubjects in pilot phase I clinical studies (Dorr et al., Life Sciences58:1777-1784 (1996)). Due to the lack of ligands specific for particularMC receptors, however, the specific receptor associated witherectogenesis has been uncertain (Vergoni et al., Eur. J. Pharmacol.362:95-101 (1998)).

The present invention discloses that the receptor MC-3 is specificallyassociated to sexual dysfunction. As discussed in Example VI, this isdemonstrated using a compound that acts as an MC-3-specific antagonist.Therefore, ligands for MC-3 that can alter the activity of an MC-3receptor can be useful for treating sexual dysfunction and otherconditions or conditions associated with MC-3 such as inflammation.

Other MC-3-associated conditions that can be treated with the MC-3receptor ligands include disuse deconditioning; organ damage such asorgan transplantation or ischemic injury; adverse reactions associatedwith cancer chemotherapy; diseases such as atherosclerosis that aremediated by free radicals and nitric oxide action; bacterial endotoxicsepsis and related shock; adult respiratory distress syndrome; andautoimmune or other patho-immunogenic diseases or reactions such asallergic reactions or anaphylaxis, rheumatoid arthritis, inflammatorybowel disease, ulcerative colitis, glomerulonephritis, systemic lupuserythematosus, transplant atherosclerosis and parasitic mediated immunedysfunctions such as Chagas's Disease.

Accordingly, the present invention provides a method for selecting anMC-3 ligand by initially contacting a compound with an MC-3 receptor. Itis then determined whether the compound modulates the activity of thereceptor. The compound is selected if the compound modulates theactivity of the receptor.

A variety of assays can be used to measure activity modulation by MC-3receptor ligands. Because MC receptors are G-protein-coupled receptorsthat activate adenylate cyclase and produce cAMP in response to bindingof ligands, a cAMP assay can be useful for determining whether acompound can modulate an MC-3 receptor's activity. Such as assay isdescribed in Example IV.

A particular MC-3 ligand can modulate the receptor's activity as anagonist. The term “agonist” means herein a ligand that increases orotherwise stimulates the activity of the MC-3 receptor. An increase inactivity can be detected in a cAMP assay by elevation in cAMP comparedto a negative control. The potency of the agonist can be represented interms of EC₅₀, which is the concentration of ligand necessary to achieve50% of the maximum increase by that agonist in an assay. Thus, an MC-3agonist can have an EC₅ less than 1 μM. More potent agonists can have anEC₅₀ less than 500, 200, 100 or 50 nM. HP-228, which is a powerful MC-3agonist, can be used as a positive control for the assay. Thus, the MC-3ligand can also be measured in comparison with HP-228, having at least25%, 50%, 75% or even 100% of the MC-3 stimulatory effect of HP-228 inthe assay.

A particular MC-3 ligand can also act as an antagonist. The term“antagonist” herein means a ligand that binds to an MC-3 receptor,resulting in any decrease in the receptor's activity. The decrease canbe determined in a cAMP assay by detecting a reduction in thestimulatory effect of HP-228. The potency of the antagonist can also berepresented in terms of EC₅, where EC₅₀ is the concentration of ligandnecessary to achieve 50% of the maximum decrease by that antagonist inan assay. An example of an antagonist is TRG 2411 #203, as described inExample II.A.

An MC-3 antagonist can be particularly useful for decreasing anMC-3-associated condition. For example, where penile erections aremediated by MC-3, decreasing the MC-3 activity using an MC-3 antagonistcan be useful for treating priapism or otherwise reducing the ability ofa subject to maintain an erection.

While a ligand that binds MC-3 and modulates MC-3 activity is useful,the same ligand may also be able to bind other MC receptors. If so, theligand may also modulate the activity of those other receptors to somedegree. While the presence of non-MC-3 activity may not necessarily bedetectable or interfere with the intended effect of an MC-3 ligand, itcan be desirable to select an MC-3 ligand so that such non-MC-3 activityis minimized. Thus, a particularly useful ligand can also showpreferential or selective activity for MC-3 compared to othermelanocortin receptors.

Thus, the selection method described above can further comprise the stepof determining whether the ligand is MC-3-preferring compared to asecond melanocortin receptor. The compound can then be selected only ifthe compound is both an MC-3 ligand and MC-3-preferring. The term“second melanocortin receptor” as used herein can be any one orcombination of MC-1, MC-2, MC-4, MC-5, or any other known melanocortinreceptor other than MC-3.

A ligand can be “MC-3-preferring” in two ways, as illustrated in ExampleV. First, an agonist can be MC-3-preferring if the ligand has a lowerEC₅₀ for MC-3 than for the second MC. For example, the ligand's EC₅ forMC-3 can be less than 5%, 10%, 20%, 50% or 100% of the ligand's EC₅₀ forthe second MC, as measured by a cAMP assay. Similarly, anMC-3-preferring antagonist can have a lower EC₅₀ for MC-3 than for thesecond MC.

A ligand agonist or antagonist can also be MC-3-preferring by bindingmore tightly to MC-3 than to another melanocortin receptor. For example,the ability of a ligand compound to compete for binding of a known MCreceptor ligand can be used to assess the affinity and specificity ofthe compound for one or more MC receptors. An example of such acompetition assay is presented in Example III.

Any MC receptor ligand can be used so long as the competing ligand canbe labeled with a detectable moiety. The detectable moiety can be aradiolabel, fluorescent label or chromophore, or any detectablefunctional moiety so long as the MC receptor ligand exhibits specific MCreceptor binding. A particularly useful detectable MC receptor ligandfor identifying and characterizing other MC receptor ligands is ¹²⁵I-HP467, which has the amino acid sequenceAc-Nle-Gln-His-(para-¹²⁵iodo-(D)Phe)-Arg-(D)Trp)-Gly-NH₂.

The binding affinity of a ligand for an MC receptor can be expressed asan IC₅₀ value, which is the concentration giving 50% inhibition ofbinding of ¹²⁵I-HP 467. Thus, an MC-3-preferring ligand can have a lowerIC₅₀ for MC-3 than for the second MC. For example, the ligand's IC₅₀ forMC-3 can be less than 5%, 10%, 20%, 50% or 100% of the ligand's IC₅₀ forthe second MC.

While an MC-3 ligand can have similar EC₅₀ and IC₅₀ profiles acrossseveral MC receptors, the profiles can also be different due todifferent receptor sources used in the assays. For example EC₅₀ profilesmay be determined using nonhuman MC receptors such as mouse or rat,while IC₅₀ profiles are determined using human MC receptors, or viceversa. Thus, both activity and binding preferences, as manifested byEC₅₀ and IC₅₀ profiles, can be separately useful for determining theMC-3 preference of a ligand. In addition, MC preferences may also differwhen the profiles are determined in vivo or in vitro.

The invention also provides a method for screening a library ofcompounds for MC-3 ligands by selecting the compounds from the libraryfor MC-3 modulating activity, as described above. The library can haveat least 50, 100, 200, 500 or even 1000 compounds. Such libraries havebeen described extensively in the literature and can be generated bycombinatorial or other methods or can be obtained commercially.

Compounds obtained by screening such libraries may not be immediatelyadministerable to a subject and may require an additionalpharmaceutically acceptable carrier, as described above. In particular,the ligand may not be readily soluble or be able to reach the intendedtarget area in effective concentrations. For example, the ligand may notreadily cross the blood-brain barrier upon administration. One skilledin the art will recognize that numerous methods are known in the art tosolubilize and administer initially insoluble compounds, such asdissolving the compound in 20% DMSO or 20% CDEX dextran (w/v). As alsodiscussed above, these solubility and other considerations will berecognized by the skilled artisan when determining the effective dose ofthe compound when used to treat an MC-3-associated condition.

The invention further provides a method for treating an MC-3-associatedcondition in a subject. The term “MC-3-associated condition” includesany condition or condition mediated by MC-3 or can be affected bybinding an MC-3 ligand. Such conditions include inflammation and sexualdysfunction. The method comprises administering to the subject aneffective dose of a pharmaceutical composition, which comprises apharmaceutically acceptable carrier and a compound obtained by screeninga compound library, as described above.

Useful compounds obtained from screening libraries includebenzamidazoles, which have the generic starting structure

The R₁, R₂ and R₃ positions can then be derivatized with compoundshaving functional groups using standard organic chemistry techniques.The variable R₁ can be derivatized with an amino acid. The variable R₂can be derivatized with a primary or secondary amine. The variable R₃can be derivatized with an aldehyde.

Particular compounds can be selected by further defining one or more ofthe individual variables in the generic formula above. Thus, R₁ can bederivatized with arginine; R₂ can be derivatized withphenethylbenzylamine or 2-phenyl-phenethylamine; and R₃ can bederivatized with 4-t-butylbenzaldehyde, 4-i-propylbenzaldehyde or4-butoxybenzaldehyde.

Specific benzamidazole compounds are designated Compounds A to E asfollows. In Compound A R₁ is derivatized with arginine, R₂ isderivatized with phenethylbenzylamine and R₃ is derivatized with4-t-butylbenzaldehyde:

In Compound B, R₁ is derivatized with arginine, R₂ is derivatized with2-phenyl-phenethylamine and R₃ is derivatized with 4-butoxybenzaldehyde:

In Compound C, R₁ is derivatized with arginine, R₂ is derivatized with2-phenyl-phenethylamine and R₃ is derivatized with4-i-propylbenzaldehyde:

In Compound D, R₁ is derivatized with arginine, R₂ is derivatized withphenethylbenzylamine and R₃ is derivatized with 4-i-propylbenzaldehyde:

In Compound E, R₁ is derivatized with arginine, R₂ is derivatized with1,2-diphenolethylamine and R₃ is derivatized with 4-t-butylbenzaldehyde:

The following examples are intended to illustrate but not limit theinvention.

EXAMPLE I Synthesis of Compounds A. Synthesis of HP-228

HP-228 was synthesized essentially as described in U.S. Pat. No.5,420,109.

Briefly, 100 mg MBHA resin containing a t-Boc Gly derivative was addedto a reaction vessel suitable for solid phase peptide synthesis(Houghten, Proc. Natl. Acad. Sci. USA 82:5131 (1985)). The followingconditions were used for peptide synthesis: coupling was performed in6-fold excess in N,N-dimethylformamide (DMF) with 0.2 MN-hydroxybenzotriazole(HOBt) and 0.2 M N,N-diisopropylcarbodiimide (DIC)for 90 minutes; activation was performed with 5% diisopropylethylamine(DIEA) in methylene chloride (DCM) for three washes of 2 min;deprotection was performed with 55% trifluoroacetic acid (TFA) for 30min; washes were performed with DCM and isopropanol; the ninhydrin testwas run after washing with DMF, DCM and methanol; acetylation wasperformed with acetylimidazole in 40-fold excess DCM for 4 hr; andcleavage was performed with hydrofluoric acid (HF) and anisole for 90min.

Peptide synthesis was carried out with the sequential steps ofactivation, coupling of amino acid, ninhydrin test, deprotection andwashing, and the steps were repeated for addition of a new amino acid ateach cycle. The amino acids were coupled in the order D-Trp, L-Arg,(D)Phe, L-His, L-Gln and L-Nle. The peptide was acetylated and the DNPprotecting group was removed from is using 2.5% thiophenol in DMFfollowed by removal of formyl protecting groups in 25% HF indimethylsulfide. The peptide was cleaved from the resin and processed asdescribed previously (U.S. Pat. No. 5,420,109). The resulting peptidewas approximately 80 to 90% pure.

B. Preparation of HP-467

Compound HP-467 was prepared by iodinating HP-228, described above. Theiodinated compound can be radiolabeled or unlabeled.

For iodination, 100 μl 2 N H₂SO₄ and 400 μl 0.5 M CuSO₄ was added to 12mg Zn powder, and the components were allowed to react with periodicmixing for 30 to 45 min, with venting, until bubbling stopped. Thegrains were washed twice with water. For unlabeled peptide, 7.12 μl of0.67 mM NaI (0.0047 μmol) was added to the reaction vial. Forradiolabeled peptide, 0.0047 μmol of Na¹²⁵I was added to the reactionvial. Approximately ⅛ of the copper grains were added to the vial, andthe vial was vortexed for 1 minute. The reaction was carried out ventedat room temperature for 3 hr with periodic mixing.

Samples were analyzed on a Vydac 218TP54 C-18 column and were monitoredat 214 nm. Buffer A was 0.05% TFA in water, and Buffer B was 0.05% TFAin acetonitrile. Samples were resolved using a 2% per minute gradientfrom 5 to 55% Buffer B in 25 min.

Using this method, ¹²⁵I-HP 467 was routinely labeled to a specificactivity of 2000 Ci/mmol. These results demonstrate that HP-467 can beiodinated to generate unlabeled iodo-peptide or high specific activityradiolabeled iodo-peptide.

EXAMPLE II Induction of Erections Upon Administration of HP-228 A.Induction of Erections in Rats

Adult male rats were housed 2 to 3 per cage and were acclimated to thestandard vivarium light cycle (12 hr. light, 12 hr. dark), rat chow andwater for a least a week prior to testing. All experiments wereperformed between 9 a.m. and noon and rats were placed in cylindrical,clear plexiglass chambers during the 60 minute observation period.Mirrors were positioned below and to the sides of the chambers toimprove viewing.

Observations began 10 minutes after an intraperitoneal injection ofeither saline or compound. An observer counted the number of groomingmotions, stretches, yawns and spontaneously occurring penile erectionsnot elicited by genital grooming, and then recorded them every 5 minutesfor a total of 60 minutes. The observer was unaware of the treatment andanimals were tested once, with n =6 in each group. Values in the figuresrepresent the group mean. HP-228 was used as a positive control forpenile erections. Significant differences between groups were determinedby an overall analysis of variance and the Student Neunmann-Keuls posthoc test was used to identify individual differences between groups (p<0.05).

Upon observation, an average of 4.8 erections were observed per hour,compared to 0.2 for vehicle (PBS), thus demonstrating that HP-228 caninduce erections in rats. Injection with HP-228 with antagonist TRG 2411#203 reduced the average number of erections observed to 2.7.

B. Dose-response Curve in Rats

This example describes a dose-response correlation for using HP-228 toinduce erections in rats.

Adult male Sprague-Dawley rats weighing 220 to 250 g or adult maleBalb/c (Harlan Laboratories; Indianapolis Ind.) were anesthetized andthen injected intracerebroventricularly (ICV) with 5 to 10 microliters(1 ml/kg body weight) HP-228 solution or PBS as a negative control.Concentrations of HP-228 were 0.45, 0.9, 1.8, 3.6 and 7.2 mg/kg (n =4 to6 animals). The animals were allowed 10 minutes to recover from theanesthesia before observation. The animals were then observed for 60 to90 minutes for penile erections not induced by grooming. Yawning,stretching, grooming behaviors were also recorded.

mg/kg HP-228 avg. erections observed/hour 0.0 (control) 0.25 0.45 2.800.9 2.60 1.8 3.67 3.6 7.00 7.2 5.75

These results demonstrate a dose-response effect by HP-228 for inducingerections in rats.

C. Dose-response Curve in Mice

This example describes a dose-response correlation for using HP-228 toinduce erections in mice (n=3 to 5). Age-matched C57/B16 mice (HarlanLaboratories; Indianapolis IN) weighing 20 to 25 g were injected (100μl/mouse) essentially as described above and observed.

μg/mouse HP-228 avg. erections observed/hour 0 (control) 0.33 25 3.00 506.67 100 5.33 250 3.00

These results demonstrate that HP-228 is also erectogenic in mice.

EXAMPLE III Melanocortin Receptor Assay

This example describes methods for assaying ligand binding to MCreceptors.

All cell culture media and reagents were obtained from GibcoBRL(Gaithersburg Md.), except for COSMIC CALF SERUM (HyClone; Logan Utah).HEK 293 cell lines were transfected with human MC receptors hMCR-1,hMCR-3 and hMCR-4 (Gantz et al., Biochem. Biophys. Res. Comm.200:1214-1220 (1994); Gantz et al., J. Biol. Chem. 268:8246-8250 (1993);Gantz et al. J. Biol. Chem. 268:15174-15179 (1993); Haskell-Leuvano etal., Biochem. Biophys. Res. Comm. 204:1137-1142 (1994)). Vectors forconstructing an hMCR-5-expressing cell line were obtained and a line ofHEK 293 cells expressing hMCR-5 was constructed (Gantz, supra, 1994).Receptor hMCR-5 has been described previously (Franberg et al., Biochem.Biophys. Res. Commun. 236:489-492 (1997); Chowdhary et al., Cytogenet.Cell Genet. 68:1-2 (1995); Chowdhary et al., Cytogenet. Cell Genet.68:79-81 (1995)). HEK 293 cells were maintained in DMEM, 25 mM HEPES, 2mM glutamine, non-essential amino acids, vitamins, sodium pyruvate, 10%COSMIC CALF SERUM, 100 units/ml penicillin, 100 pg/ml streptomycin and0.2 mg/ml G418 to maintain selection.

Before assaying, cells were washed once with phosphate buffered saline(PBS without Ca²+and Mg²⁺) and stripped from the flasks using 0.25%trypsin and 0.5 mM EDTA. Cells were suspended in PBS, 10% COSMIC CALFSERUM and 1 mM CaCl₂. Cell suspensions were prepared at a density of2×10⁴ cells/ml for HEK 293 cells expressing hMCR-3, hMCR-4 or hMCR-5,and 1×10⁵ cells/ml for HEK 293 cells expressing hMCR-1. Suspensions wereplaced in a water bath and allowed to warm to 37° C for 1 hr.

Binding assays were performed in a total volume of 250 μl for HEK 293cells. Control and test compounds were dissolved in distilled water.Custom labeled ligand ¹²⁵I-HP 467 (50,000 dpm, 2000 Ci/mmol) (Amersham;Arlington Heights Ill.) was prepared in 50 mM Tris, pH 7.4, 2 mg/ml BSA,10 mM CaCl₂, 5 mM MgCl₂, 2 mM EDTA and added to each tube. To each tubewas added 4×10³ HEK 293 cells expressing hMCR-3, hMCR-4 or hMCR-5, or2×10⁴ cells expressing hMCR-1. Assays were incubated for 2.5 hours at37° C.

GF/B filter plates were prepared by soaking for at least one hour in 5mg/ml BSA and 10 mM CaCl₂. Assays were filtered using a Brandel 96-wellcell harvester (Brandel Inc.; Gaithersburg Md.). The filters were washedfour times with cold 50 mM Tris, pH 7.4; the filter plates weredehydrated for 2 hr; and 35 μl of MICROSCINT was added to each well.Filter plates were counted using a Packard Topcount (Packard InstrumentCo.; Meriden Conn.) and data analyzed using GraphPad PRISM v2.0(GraphPad Software Inc.; San Diego Calif.) and Microsoft EXCEL v5.0a(Microsoft Corp.; Redmond Wash.).

To assay compounds, binding assays were performed in duplicate in a96-well format. HP 467 was prepared in 50 mM Tris, pH 7.4, and ¹²⁵I-HP467 was diluted to give 100,000 dpm per 50 μl. A compound was added tothe well in 25 μl aliquots. A 25 μl aliquot of ¹²⁵I-HP 467 was added toeach well. A 0.2 ml aliquot of suspended cells was added to each well togive the cell numbers indicated above, and the cells were incubated at37° C. for 2.5 hours. Cells were harvested on GF/B filter plates asdescribed above and counted.

EXAMPLE IV cAMP Assay for Melanocortin Receptors

This example describes methods for assaying cAMP production fromG-protein-coupled MC receptors.

HEK 293 cells expressing MCR-1, MCR-3, MCR-4 and MCR-5 were used. Cellswere plated at 20,000 cells per well in a 96-well plate coated withcollagen. The next day, cells were pretreated with 75 μl of 0.4 mM3-isobutyl-1-methylxanthine (IBMX) in low serum medium containing DMEM,25 mM HEPES, non-essential amino acids, vitamins, 100 units/mlpenicillin, 100 μg/ml streptomycin and 0.1% COSMIC CALF SERUM. IBMX isan inhibitor of cAMP phosphodiesterase. The pretreatment was carried outfor 10 minutes at 37° C.

Following pretreatment, 25 μl of diluted compound was added to the wellsand cells were incubated for 15 min at 37° C. Cells were lysed by adding25 μl saponin lysis buffer and incubating 2 to 5 minutes. Plates werecovered and stored at −20° C.

The cAMP concentration was determined by ELISA. Briefly, 96 well ELISAplates were coated with goat anti-cAMP antibody in PBS for 12 to 72 hrat 4° C. 50 μl of sample was mixed with 50 μl of cAMP ELISA buffercontaining 1% bovine serum albumin, 10% heat inactivated donor horseserum, 1% normal mouse serum and 0.05% TWEEN-20 in PBS, and the dilutedsample was added to the coated ELISA plate. Standards of knownconcentrations of cAMP were added to separate wells. Then,cAMP-conjugated horseradish peroxidase (cAMP-HRP) was added to each wellat 25 μl of 16 ng/ml. The plates were incubated for 3 hr at roomtemperature. Plates were washed and the binding of cAMP-HRP was detectedwith 3,3′,5,5′-tetramethylbenzidine (TMB) and hydrogen peroxide usingstandard immunoassay procedures.

EXAMPLE V Melanocortin Receptor Binding Profile of Compounds

Various compounds were tested for in vitro activity and binding tomelanocortin receptors. MC-3 stimulatory effect is also described interms of percentage of stimulation obtained by adding HP-228. The EC₅₀values are the concentration of ligand necessary to achieve 50% of themaximum increase by that ligand in an assay. The IC₅₀ values are theconcentration giving 50% inhibition of binding of ¹²⁵I-HP 467.

EC₅₀ (μM) values Compound % of HP-228 MC-3 MC-1 MC-4 A 89% 7.65 0.47 B115% 1.33 12 C 92% 1.4 10.6 D 140% 1.7 2.8 E 59% 6.7 0.43 19.9

These results show that the Compounds A to E can act as MC-3 agonists,often exceeding the stimulatory activity of HP-228 in comparable assays.Moreover, Compounds B to E each have a greater stimulatory effect onMC-3 than another MC receptor, thus demonstrating MC-3-preferringactivity.

Compounds E and F (structures not shown) were tested for competitivebinding to MC-3, MC-1, MC-4 and MC-5.

IC₅₀ (μM) values Compound MC-3 MC-1 MC-4 MC-5 F 2.664 1.668 2.849 0.343G 4.015 1.175 5.808 0.325 H 1.455 0.645 4.682 0.269 I 0.405 3.385 2.1640.172 J 10.863 15.496 20.160 28.049

These results demonstrate that compounds such as Compound J can havestronger binding to MC-3 compared to other melanocortin receptors. Inaddition, although Compound I does binds MC-5, it binds MC-3 moretightly than MC-1 or MC-4 and is therefore considered MC-3-preferring.

EXAMPLE V Association of MC-3 With Erectile Response

This example shows that erectogenesis is associated with MC-3.

Sprague-Dawley rats (n=5) received 1.8 mg/kg HP-228 or PBS via IP. Therats also received via ICV a dose of PBS or MC-3-specific antagonist at10 μg/5 μl. After administration, the rats were observed as describedabove.

In the negative control rats, receiving only PBS, 0.3 erections per hourwere observed. In the positive control rats, receiving HP-228 but noMC-3 antagonist, 3.8 erections per hour were observed. In rats receivingHP-228 and the MC-3-specific antagonist, the number of erectionsobserved decreased to 0.8 per hour, demonstrating that theHP-228-induced erections were antagonized by the MC-3-specificantagonist, thereby identifying MC-3 as the MC receptor associated witherectogenesis. No erections were observed in the rats receiving theMC-3-specific antagonist and no HP-228. The MC-3-specific antagonist isalso known to have MC-4 agonist properties, which were reflected bysignificantly reduced food consumption.

Although the invention has been illustrated by the examples above, itshould be understood that various modifications can be made withoutdeparting from the spirit of the invention. Accordingly, the inventionis limited only by the following claims.

We claim:
 1. A method for treating erectile dysfunction in a malesubject, comprising the step of administering to the subject aneffective dose of the compound X₁-X₂-(D)Phe-Arg-(D)Trp-X₃ wherein

 COCH₃, H or absent;

 His, COCH₃ or H; and

 or R₅; wherein R₁ is H, COCH₃, C₂H₅, CH₂Ph, COPh, COO-t-butyl,COOCH₂Ph, CH₂CO-(polyethylene glycol) or A; R₂ is H, COCH₃, C₂H₅ orCH₂Ph; R₃ is a linear alkyl group having 1 to 6 carbon atoms or abranched or cyclic alkyl group having 3 to 6 carbon atoms; R₄ is(CH₂)_(m)—CONH₂, (CH₂)_(m)—CONHR₁ or (CH₂)_(m)—CONHA; R₅ is OH, OR₃,NH₂, SH, NHCH₃, NHCH₂Ph or A; R₆ is H or R₃; R₇ is H, COCH₃, C₂H₅,CH₂Ph, COPh, COO-t-butyl, COOCH₂Ph or CH₂CO-(polyethylene glycol); Ph isC₆H₅; m is 1, 2 or 3; n is 0, 1, 2 or 3; Y¹ and Y² are independentlyhydrogen atoms, or are taken together to form a carbonyl orthiocarbonyl; and A is


2. The method of claim 1, wherein X₁ is selected from the groupconsisting of norleucine and Ac-norleucine.
 3. The method of claim 1,wherein X₁ is selected from the group consisting of norvaline,Ac-norvaline, leucine, Ac-leucine, isoleucine and Ac-isoleucine.
 4. Themethod of claim 1, wherein X₂ is selected from the group consisting ofGln-His and His.
 5. The method of claim 1, wherein X₃ is selected fromthe group consisting of Gly and Gly-NH₂.
 6. The method of claim 1,wherein R₁ is selected from the group consisting of H, C₂H₅ and CH₂Ph.7. The method of claim 1, wherein R₁ and R₂ are independently selectedfrom the group consisting of COCH₃ and H.
 8. The method of claim 1,wherein R₅ is NH₂.
 9. The method of claim 1, wherein R₅ is covalentlybound to X₁, forming a cyclic peptide.
 10. The method of claim 1,wherein the compound HP-228, having the structureAc-Nle-Gln-His-(D)Phe-Arg-(D)Trp-Gly-NH₂.
 11. The method of claim 1,wherein the (D)Phe is iodinated in the para position.
 12. The method ofclaim 11, wherein the compound isAc-Nle-Gln-His-(para-iodo-(D)Phe)-Arg-(D)Trp-Gly-NH₂.
 13. The method ofclaim 1, wherein the compound is selected from group consisting of(D)Phe-Arg-(D)Trp, Ac-(D)Phe-Arg-(D)Trp, (D)Phe-Arg-(D)Trp-NH₂ andAc-(D)Phe-Arg-(D)Trp-NH₂.
 14. The method of claim 1, wherein thecompound is selected from group consisting of(cyclohexyl)Gly-Gln-His-(D)Phe-Arg-(D)Trp-Gly,Ac-(cyclohexyl)Gly-Gln-His-(D)Phe-Arg-(D)Trp-Gly-NH₂ and cyclo(His-(D)Phe-Arg-(D)Trp).
 15. The method of claim 1, wherein the compoundis selected from group consisting ofNle-Gln-His-(D)Phe-Arg-(D)Trp-Gly-NH₂, His-(D)Phe-Arg-(D)Trp-Gly,His-(D)Phe-Arg-(D)Trp-Gly-NH₂, Ac-His-(D)Phe-Arg-(D)Trp-NH₂,His-(D)Phe-Arg-(D)Trp-OH, His-(D)Phe-Arg-(D)Trp,His-(D)Phe-Arg-(D)Trp-NH₂, Ac-His-(D)Phe-Arg-(D)Trp-OH andAc-His-(D)Phe-Arg-(D)Trp-Gly-NH₂.
 16. The method of claim 1, wherein thecompound is selected from group consisting ofAc-Nle-Gln-His-(D)Phe-Arg-(D)Trp-Gly-OH,Ac-Nle-Gln-His-(D)Phe-Arg-(D)Trp-Gly-OC₂H₅,Ac-Nle-Gln-His-(D)Phe-Arg-(D)Trp-Gly-NH-NH₂,Ac-Nle-Asn-His-(D)Phe-Arg-(D)Trp-Gly-NH₂,Ac-Nle-Asn-His-(D)Phe-Arg-(D)Trp-Gly-OH,Ac-Nle-Gln-His-(D)Phe-Arg-(D)Trp-Gly-NHCH₂CH₂Ph,Ac-Nle-Gln-His-(D)Phe-Arg-(D)Trp-Gly-NHCH₂Ph,

Gln-His-(D)Phe-Arg-(D)Trp-Gly-NH₂, Ac-Gln-His-(D)Phe-Arg-(D)Trp-Gly-NH₂,Ac-Nle-Gln-His-(D)Phe-Arg-(D)Trp-NH₂,Ac-His-(D)Phe-Arg-(D)Trp(CH₂)-(NAc)Gly-NH₂ andHis-(D)Phe-Arg-(D)Trp(CH₂)-(NAc)Gly.
 17. A method for treating sexualdysfunction in a female subject, comprising the step of administering tothe subject an effective dose of the compound X₁-X₂-(D)Phe-Arg-(D)Trp-X₃wherein

 COCH₃, H or absent;

 His, COCH₃ or H; and

 or R₅; wherein R₁ is H, COCH₃, C₂H₅, CH₂Ph, COPh, COO-t-butyl,COOCH₂Ph, CH₂CO-(polyethylene glycol) or A; R₂ is H, COCH₃, C₂H₅ orCH₂Ph; R₃ is a linear alkyl group having 1 to 6 carbon atoms or abranched or cyclic alkyl group having 3 to 6 carbon atoms; R₄ is(CH₂)_(m)—CONH₂, (CH₂)_(m)—CONHR₁ or (CH₂)_(m)—CONHA; R₅ is OH, OR₃,NH₂, SH, NHCH₃, NHCH₂Ph or A; R₆ is H or R₃; R₇ is H, COCH₃, C₂H₅,CH₂Ph, COPh, COO-t-butyl, COOCH₂Ph or CH₂CO-(polyethylene glycol); Ph isC₆H₅; m is 1, 2 or 3; n is 0, 1, 2 or 3; Y¹ and Y² are independentlyhydrogen atoms, or are taken together to form a carbonyl orthiocarbonyl; and A is


18. The method of claim 17, wherein X₁ is selected from the groupconsisting of norleucine and Ac-norleucine.
 19. The method of claim 17,wherein X₁ is selected from the group consisting of norvaline,Ac-norvaline, leucine, Ac-leucine, isoleucine and Ac-isoleucine.
 20. Themethod of claim 17, wherein X₂ is selected from the group consisting ofGln-His and His.
 21. The method of claim 17, wherein X₃ is selected fromthe group consisting of Gly and Gly-NH₂.
 22. The method of claim 17,wherein R₁ is selected from the group consisting of H, C₂H₅ and CH₂Ph.23. The method of claim 17, wherein R₁ and R₂ are independently selectedfrom the group consisting of COCH₃ and H.
 24. The method of claim 17,wherein R₅ is NH₂.
 25. The method of claim 17, wherein R₅ is covalentlybound to X₁, forming a cyclic peptide.
 26. The method of claim 17,wherein the compound HP-228, having the structureAc-Nle-Gln-His-(D)Phe-Arg-(D)Trp-Gly-NH₂.
 27. The method of claim 17,wherein the (D)Phe is iodinated in the para position.
 28. The method ofclaim 27, wherein the compound isAc-Nle-Gln-His-(para-iodo-(D)Phe)-Arg-(D)Trp-Gly-NH₂.
 29. The method ofclaim 17, wherein the compound is selected from group consisting of(D)Phe-Arg-(D)Trp, Ac-(D)Phe-Arg-(D)Trp, (D)Phe-Arg-(D)Trp-NH₂ andAc-(D)Phe-Arg-(D)Trp-NH₂.
 30. The method of claim 17, wherein thecompound is selected from group consisting of(cyclohexyl)Gly-Gln-His-(D)Phe-Arg-(D)Trp-Gly,Ac-(cyclohexyl)Gly-Gln-His-(D)Phe-Arg-(D)Trp-Gly-NH₂ andcyclo(His-(D)Phe-Arg-(D)Trp).
 31. The method of claim 17, wherein thecompound is selected from group consisting ofNle-Gln-His-(D)Phe-Arg-(D)Trp-Gly-NH₂, His-(D)Phe-Arg-(D)Trp-Gly,His-(D)Phe-Arg-(D)Trp-Gly-NH₂, Ac-His-(D)Phe-Arg-(D)Trp-NH₂,His-(D)Phe-Arg-(D)Trp-OH, His-(D)Phe-Arg-(D)Trp,His-(D)Phe-Arg-(D)Trp-NH₂, Ac-His-(D)Phe-Arg-(D)Trp-OH andAc-His-(D)Phe-Arg-(D)Trp-Gly-NH₂.
 32. The method of claim 17, whereinthe compound is selected from group consisting ofAc-Nle-Gln-His-(D)Phe-Arg-(D)Trp-Gly-OH,Ac-Nle-Gln-His-(D)Phe-Arg-(D)Trp-Gly-OC₂H₅,Ac-Nle-Gln-His-(D)Phe-Arg-(D)Trp-Gly-NH-NH₂,Ac-Nle-Asn-His-(D)Phe-Arg-(D)Trp-Gly-NH₂,Ac-Nle-Asn-His-(D)Phe-Arg-(D)Trp-Gly-OH,Ac-Nle-Gln-His-(D)Phe-Arg-(D)Trp-Gly-NHCH₂CH₂Ph,Ac-Nle-Gln-His-(D)Phe-Arg-(D)Trp-Gly-NHCH₂Ph,

Gln-His-(D)Phe-Arg-(D)Trp-Gly-NH₂, Ac-Gln-His-(D)Phe-Arg-(D)Trp-Gly-NH₂,Ac-Nle-Gln-His-(D)Phe-Arg-(D)Trp-NH₂,Ac-His-(D)Phe-Arg-(D)Trp(CH₂)-(NAc)Gly-NH₂ andHis-(D)Phe-Arg-(D)Trp(CH₂)-(NAc)Gly.
 33. The method of claim 17, whereinthe dysfunction is sexual arousal disorder.